Accumulating studies highlight a critical role for endothelial cell (EC) inflammation and dysfunction in the pathogenesis of septic shock and sepsis-induced lung injury, leading causes of death among critically ill patients. Excessive generation of pro-inflammatory mediators can lead to collateral vascular dysfunction, an effect that confers pro-adhesive, pro-permeability, and pro-thrombotic properties to ECs. Therefore, modulating these events in the vascular endothelium may provide a novel therapeutic approach to limit the sequelae of sepsis and sepsis-induced lung injury. MicroRNAs (miRNAs) are small, non-coding RNAs that suppress the expression of target genes at the post-transcriptional level and are involved in a range of biological responses. However, the role of microRNAs in sepsis-associated endothelial dysfunction remains poorly defined. Using a microarray profiling approach in ECs, we identified that miR-181b expression is rapidly reduced in the vascular endothelium from endotoxemic mice - observations that are recapitulated in human subjects with sepsis in vivo. Our studies have uncovered that miR-181b inhibits targets that control 2 key signaling pathways, NF-kB and AKT/eNOS, that govern EC adhesion, vascular permeability, and proinflammatory mediators implicated in sepsis and sepsis-induced lung injury. Preliminary functional studies in ECs reveal that miR-181b potently inhibits effects on leukocyte adhesion, EC permeability, and thrombin-induced EC inflammation. MiR-181b suppresses the activation of the NF-kB pathway uniquely in ECs by binding to the 3'UTR of importin-a3, a protein involved in NF-kB nuclear translocation in ECs and not leukocytes. In contrast, miR-181b induces eNOS-phosphorylation by directly targeting the phosphatase PHLPP2, known to inhibit AKT-phosphorylation. Finally, treatment of mice by systemic intravenous administration of miR-181b mimics as replacement therapy reduces endotoxemia-induced EC inflammatory markers, leukocyte accumulation, lung injury, and markedly improves survival. Thus, we hypothesize that miR-181b may serve as a critical homeostatic regulator of ECs and vascular dysfunction in sepsis and sepsis-induced lung injury. To further understand the protective role of miR-181b in sepsis, we propose: in Aim1, to delineate the proximal events during sepsis regulating miR-181b expression. In Aim2, using inflammatory stimuli including human plasma samples from patients with sepsis and sepsis-induced lung injury, we will dissect the mechanisms by which miR-181b regulates NF-kB and AKT signaling in response to EC dysfunction. In Aim3, we will explore the effect and timing of altered miR-181b expression, or its targets (using genetic, siRNA, or pharmacological approaches to importin-a3 or PHLPP2), on experimental models of sepsis and EC function in vivo. Successful completion of the proposed studies will identify significant insights regarding miR-181b function in EC inflammation, vascular leak, and microvascular thrombosis, and may provide discrete, novel targets for sepsis-induced lung injury.